Working Group on Coagulation Standards
DRAFT Subcommittee Minutes
24 July 2011
12:30-2:30
Room B-2
Chairman: Anthony Hubbard
Review of Lot #3 (A Hubbard)
Between July 2010 and end June 2011 a total of 8,840 vials of Lot #3 were issued. Orders were despatched to 21 different manufacturers and 1 external quality assurance scheme. Lot #3 was also used in two collaborative studies (calibration of Lot #4 and the calibration of von Willebrand factor propeptide). Since the issue of Lot #3 commenced in 2006 approximately 48,000 vials have been despatched – this leaves remaining stock of approximately 6,700 vials. At the current rate of issue it is expected that Lot #3 stocks will be exhausted in Q2 2012. The stability testing of Lot #3 comprised both accelerated degradation testing and real-time studies – both indicated excellent stability with predicted mean loss of <0.1 % per year for all of the four test analytes (FV, FVII, FVIII, FIX). The results of this study have been published as an Official Communication of the ISTH (AR Hubbard, S Kitchen, M Beeharry, SA Bevan, A Bowyer. Long-term Stability of the Scientific and Standardization Committee Secondary Coagulation Standard (SSC Lot no. 3), J Thromb Haemost 2011; 9: 1246-8).
Experience of EQA schemes with Lot #3
UK NEQAS (S Kitchen)
Eight vials of SSC Lot #3 were used for “trouble-shooting” purposes between May 2010 and June 2011 relating to assay issues for Protein C activity, Antithrombin and factors II, V and VIII. Use of Lot #3 by one laboratory revealed that their discrepancy for Protein C activity estimates was linked to the routine reference plasma. After re-calibration, their difference from the mean was reduced from 9% to only 3%. A similar improvement was also found for estimates of factor V in a different laboratory where the difference from the median was reduced from 16-19% to 5-9%. Dr Kitchen then reviewed the results from NEQAS surveys based on reference plasmas with the understanding that many laboratories use reference plasmas and reagents from the same manufacturer hence it is not possible to distinguish between differences caused by the reference plasma or the reagents. Some manufacturers had not yet calibrated reference plasmas for FXI or FV relative to the WHO International Standards. The largest differences of around 20% were found between reference plasmas for FV and FVIII, however, some of this difference could be caused by the use of different reagents.
College of American Pathologists (J Teruya)
SSC Lot #3 has been issued in two surveys in the past year – one for VWF estimation and one thrombophilia survey. Dr Teruya presented the data in terms of methods-specific bias from the assigned value. A bias of greater than 10% was found for some methods for the following analytes: FVIII, Protein C activity (clotting 20%; chromogenic 11%), Protein S activity and Protein S total antigen. One clotting method for Protein S activity has produced a 20% bias in surveys performed over the last 3 years. Inter-laboratory variability did not exceed a CV of 20% for any analytes tested in 2011.
Progress towards Lot #4
Results of the calibration exercise (E Gray)
Dr Gray presented the results for the calibration of 20 analytes relative to the relevant WHO International Standards (Table 1). One new analyte (FXIII antigen) was included in the calibration of SSC Lot #4. The number of laboratories in the calibration exercises ranged from 8 to 29 and inter-laboratory variability (GCV) ranged from 2.3% (Antithrombin function) to 14.2% (VWF:Ristocetin cofactor). All participants in the collaborative studies and members of the Executive Board of the Working Group have agreed with the proposed assigned potencies. Final approval of the assigned values will be sought in the SSC Business Meeting on 27 July 2011. SSC Lot #3 was also included in the calibration exercise to enable a comparison of estimates obtained in the present study and the original calibration of Lot #3 in 2005. No significant difference (p>0.05) was found for 14 out of 19 analytes. For 4 analytes the significant difference was associated with a difference between means of 6% or less. For Protein S free antigen there was a difference of 8% which was probably associated with replacement of the primary WHO standard since the original calibration. Overall there was good agreement between the original calibration and the present study consistent with the excellent stability record for SSC Lot #3.
Lot #4 stability testing (A Hubbard)
The stability of Lot #4 will be assessed through an acceleration degradation study. Vials have been stored at elevated temperatures since December 2009 and the first tests were carried out in February 2011. Four analytes (FV, FVII, FVIII, Antithrombin activity) were tested by NIBSC and the Royal Hallamshire Hospital, Sheffield, UK. The relative residual activities after storage at +45 C indicated greatest loss for FV and FVIII, however, the calculated predictions of loss were contradictory indicating greater stability for these two analytes compared to FVII and Antithrombin. The robustness of the predictions was compromised by the small amount of data and the limited loss of activity for FVII and Antithrombin. It was agreed that more testing should be performed in Q4 2011 in order to provide more reliable predictions on which a shelf-life for Lot #4 could be based.
Table 1 Proposed assigned values for SSC Lot #4
|
Analyte |
Value (IU/vial) |
Inter-lab variability (GCV%) |
n |
|
Fibrinogen |
2.79 mg/ml |
3.8 |
22 |
|
Factor II function |
0.91 |
2.6 |
28 |
|
Factor V function |
0.89 |
5.7 |
22 |
|
Factor VII function |
0.97 |
4.1 |
29 |
|
Factor VIII function |
0.88 |
4.2 |
27 |
|
Factor IX function |
1.05 |
3.4 |
29 |
|
Factor X function |
0.94 |
2.4 |
26 |
|
Factor XI function |
0.89 |
5.2 |
20 |
|
Factor XIII function Antigen(A2B2 complex) |
0.76 0.74 |
4.6 7.6 |
17 13 |
|
von Willebrand Factor antigen ristocetin co-factor collagen binding |
1.16 0.84 1.08 |
5.9 14.2 6.4 |
22 12 8 |
|
Protein C Function antigen |
0.92 0.94 |
3.6 6.9 |
24 13 |
|
Protein S Function Free antigen Total antigen |
0.81 0.98 0.93 |
7.0 6.8 3.8 |
14 17 12 |
|
Antithrombin Function Antigen |
0.92 0.93 |
2.3 4.4 |
26 12 |
